Pivoting pressurized single use bioreactor

ABSTRACT

Pressurized hermetically sealed bags disposed inside a cylindrical support and containing a septum with variable density of porosity and dividing the bag into two chambers are used to provide optimal mixing and gasification of nutrient media to grow a variety of biological cultures, particularly the cell cultures to produce a multitude of pharmaceutical and biotechnology products in a disposable system.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.13/429,365, now U.S. Pat. No. 9,284,521 entitled “Pivoting PressurizedSingle-Use Bioreactor” filed on Mar. 24, 2012, the contents of which areincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present disclosure relates to pressurized hermetically sealed bagscontaining products used in the pharmaceutical and biotechnologyprocessing industries and, more particularly, to disposable cell bags orbioreactors.

BACKGROUND OF THE INVENTION

The bioprocessing industry has traditionally used stainless steelsystems and piping in manufacturing processes for fermentation and cellculture. These devices are designed to be steam sterilized and reused.Cleaning and sterilization are however costly labor-intensiveoperations. Moreover, the installed cost of these traditional systemswith the requisite piping and utilities is often prohibitive.Furthermore, these systems are typically designed for a specificprocess, and cannot be easily reconfigured for new applications. Theselimitations have led to adoption of a new approach over the last tenyears—that of using plastic, single-use disposable bags and tubing, toreplace the usual stainless steel tanks.

In particular bioreactors, traditionally made of stainless steel, havebeen replaced in many applications by disposable bags, which are rockedto provide the necessary aeration and mixing necessary for cell culture.These single-use bags are typically provided sterile and eliminate thecostly and time-consuming steps of cleaning and sterilization. The bagsare designed to maintain a sterile environment during operation therebyminimizing the risk of contamination.

Commonly used bags are of the “pillow style,” mainly because these canbe manufactured at low cost by seaming together two flexible sheets ofplastic.

One of the successful disposable bioreactor systems uses a rocking tableon to which a bioreactor bag is placed. The bioreactor bag is partiallyfilled with liquid nutrient media and the desired cells. The table rocksthe bag providing constant movement of the cells in the bag and alsoaeration from the turbulent air-liquid surface. The bag, typically, hasa gas supply tube for the introduction of air or oxygen, and an exhaustgas tube to allow for the removal of respired gases. Nutrients can beadded through other tubes.

One possible limitation of this type of device is that it may bedifficult to scale up beyond a few hundred liters because poor liquidcirculation causes nutrient and waste gradients that inhibit cellperformance. This is because the back and forth motion of thesingle-axis rocker used in these applications creates good liquidcirculation in the direction perpendicular to the rocking axis, butrelatively little mixing in the direction parallel to the rocking axis.In large volume bags (greater than 100 liters), or in bags with a largelength to width ratio, this poor axial circulation can result in a longtime to achieve homogeneity of the bag contents. This makes pH controlin the bioreactor bag difficult, since additions of acid or base addedto the bioreactor to modulate the pH can take a long time to dispersethroughout the bag. Nutrients added to the bioreactor bag may not bedistributed uniformly. Poor liquid circulation also limits the amount ofoxygen that can be transferred from the headspace, and thus the maximumconcentration of cells that cannot be cultured.

Circulation flow can be improved by incorporating a second axis ofrotation. By synchronizing the two axes it is possible to impart agyratory motion that greatly improves mixing and mass transfer. However,the addition of second axis increases the cost tremendously, and theincrease in mechanical complexity makes the rocker less reliable andmore difficult to maintain.

However, all of the above limitations are present U.S. Pat. No.6,190,913 to Vijay Singh (and the resultant Wave Bioreactor marketed byGE Healthcare); as a result, the same inventor proposed in U.S. patentapplication Ser. No. 12/676,180 (assigned to GE Healthcare) filed Sep.15, 2008 to overcome these limitations by creating flexible bafflesinside the bag to create a swirling motion in the bag to improve mixing.However, this modification of the existing art does not remove the mostsignificant drawback of producing stress on the seams of the bag thatprevent the use of large-scale production using these bags. The proposedmodification also does not provide complete mixing as desired from alldirections in the bag since the bag is rocked only in one dimension.

Therefore, there is a need for an apparatus that enables a user to scaleup the mixing of nutrient media in a bioreactor bag in a flexiblecontainer that will assure complete mixing nutrient media from alldirections.

SUMMARY OF THE INVENTION

The present invention has been accomplished in view of theabove-mentioned technical background, and it is an object of the presentinvention to provide a bioreactor bag that enables a user to scale upthe mixing of nutrient media and efficiently mix the nutrient media inthe bioreactor bag.

A bioreactor including a cylindrical support, a flexible containerincluding a septum that defines two chambers in the flexible containerwith the septum having such variably of porosity that the nutrient mediafrom all sides of the bag passed through the middle of the bag by usinga means to pivot the cylindrical support between −110 to +110 degrees;this causes the septum to lift the nutrient media from all sides of thebag and drain it in the middle center of the bag; reversing the cycle inan opposite direction provides the most optimal mixing of the nutrientmedia. Most efficient movement of nutrient media is observed when thecontainer has been pressurized to form a fixed volume inside so that thebioreactor acts like a hard-walled container yet it retains theadvantages of using flexible disposable containers for the manufacturingof biological products.

The container walls and the septum are flexible sheets seamed together.The two outer layers are impermeable while the septum has variableporosity creating a fluid communication between the two chambers. Theseptum has the highest porosity in the center adjacent to one of theedges. Alternatively, the container may be a modeled structure.

The container is disposed immovably in the cylindrical support such thatthe septum is vertically aligned and the highest density of pores is inthe top middle portion (top vertical and middle horizontal axes). As thecylindrical support is pivoted, the septum sweeps the liquid upwards inthe direction of movement of cylindrical support lifting the liquid andpouring it through pores along one side in the center of the septumcausing the liquid from all directions of the container to mixuniformly; repeating the operation in opposite direction on a continuousbasis produces a highly efficient and uniform mixing of the entireliquid in the container.

Notably absent in the present invention is a means of sparging orintroducing a gas in the nutrient media; the mixing of the gas with thenutrient media is achieved through surface intake into nutrient media asit is vigorously turned over repeatedly. While this means ofgasification of media is highly suitable for cell culture growth, thismay not be sufficient to support growth of bacterial cultures or thosecultures requiring very high degree of gasification.

BRIEF DESCRIPTION OF DRAWINGS

These and other advantages of the present invention will become moreapparent as the following description is read in conjunction with theaccompanying drawings, wherein:

FIG. 1A is the front view a cylindrical support containing a pressurizedflexible bag and nutrient media.

FIG. 1B is a view of the cylindrical support moved clockwise halfway.

FIG. 1C is a view of the cylindrical support moved clockwise to 110degrees.

FIG. 2A is a front view a cylindrical support containing anun-pressurized flexible bag and nutrient media.

FIG. 2B is a front view of the cylindrical support moved clockwisehalfway.

FIG. 2C is a front view of the cylindrical support moved clockwise to110 degrees.

FIG. 3A is a front view a cylindrical support containing anun-pressurized flexible bag and nutrient media.

FIG. 3B is a front view of the cylindrical support moved clockwisehalfway.

FIG. 3C is a front view of cylindrical support moved counter-clockwiseto 110 degrees.

FIG. 4 is a side view of the flexible bag vertically disposed showingthe configuration of the pores in the septum.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The presently preferred embodiments of the invention are described withreference to the drawings, where like components are identified with thesame numerals. The descriptions of the preferred embodiments areexemplary and are not intended to limit the scope of the invention.

A prior art bag is a flat, rectangular, “pillow-style” cell culture bagcommonly used in rocking bioreactor applications, for example in thesystem of U.S. Pat. No. 6,190,913 entitled “Method for Culturing CellsUsing Wave-Induced Agitation” filed Aug. 12, 1998. This prior art andother prior art bags are shown in FIG. 1-11 in the U.S. patentapplication Ser. No. 12/676,180. Another prior art bag is, which ishereby incorporated by reference.

The bag in the present invention (FIG. 1) is formed by seaming togethertwo outer flexible impervious sheets 8 and a perforated septum 7 thathas much higher porosity 6 in the center part adjacent to one of theedges forming two chambers capable of holding nutrient media 15 andresulting in an outline seam 5 that goes around the four sides of thebag and forms the point where the flexible bag is attached to the innersurface of the cylindrical support making the bag immovable. Alsoprovided are gas ports 4 attached to sterilizing filters 3 to avoid anycontamination from the outside. A liquid port 13 with a sterilizingfilter 12 is provided to introduce nutrient media that can also be usedto remove liquid from the bag after disconnecting filter 12 and also tointroduce a biological culture without the use of the filter. The bag 8is disposed inside a hard walled cylindrical support 1, which is in turncovered by a heating element 2. The cylindrical support 1 is supportedby a platform 11 through a rolling mechanism 14 that is controlled by amotor 10 through a shaft 9.

FIG. 1 has three configurations of the invention; FIG. 1A is thestarting position wherein the bag 8 is vertically disposed. Nutrientmedia 15 is introduced in the bag through port 13 and it crosses theseptum 7 since it is porous to provide an even level on both sides ofthe septum 7. It is important to note that the porosity of sheet 8 whendisposed vertical in FIG. 1a is much smaller than in the top centerportion by at least a factor of ten.

The reason for providing porosity throughout the septum is to reduce thepressure on the septum 7 when it is pivoted, allowing it to pass thenutrient media across the two chambers formed by the septum 7. It isfurther noted that in FIG. 1A, the walls of the bag completely line thehard wall of the cylindrical support and this is achieved by introducinga gas through one of the gas ports 4 to pressurize the bag. This alsocauses the bag to acquire a three-dimensional container. This wouldgenerally be desirable when using the invention to grow mammalian cellssuch as Chinese Hamster Ovary cells wherein approximately 5% carbondioxide mixed in air is provided and the mode of entry of carbon dioxidein the nutrient media is through the surface of the nutrient media.

FIG. 1 shows a clockwise movement of the cylindrical support and FIG. 1Bshows the position of the septum 7 when it is pivoted to approximately30-35 degrees. Note that the septum 7 lifts the nutrient media and whenthe rotation reaches to above 90 degrees, it drained through the septummainly through the higher porosity section 6 causing the movement ofliquid from the left chamber to the right as shown in FIG. 1A. Theoperation of the cylindrical support is then repeated in an oppositedirection to provide mixing from the right chamber to the left chamberand also from back to front. It is noteworthy that as the cylindricalsupport is pivoted, it is likely that nutrient media leaving one port toexhaust would cover one of the gas ports. Since one of the gas ports isused for introducing gas and the other used as an exhaust, the operationwill not be affected regardless of which gas port is temporarilyinundated by the nutrient media.

FIG. 2 shows a side view of the present invention disposed in a startingposition showing the septum 7 with its variable perforations 6; it isnoteworthy that the largest openings in the septum 7 are provided in thetop center position of the septum 7 when vertically disposed in thestarting position. This configuration allows the nutrient media to flowfrom the left to right and also from the back to front as the septum 7is pivoted lifting the nutrient media.

The bag 7 can be a molded three-dimensional structure or fabricated byseaming flexible sheets. The edges and gusset may be curved seams, ormanufactured as a series of straight-line seam segments as shown herein.

This invention provides an apparatus that enables a user to scale up themixing of nutrient media in a bioreactor bag. This apparatus makes itsimple to control the pH where the addition of acid or base to thebioreactor bag does not take a long time to obtain. The uniform mixingfrom the left to right and from the back to front provides an idealmixing pattern required for optimal growth of a variety of biologicalcultures. Thus, this invention provides the user with a simple method toscale up the mixing of nutrient media in a bioreactor bag to provide thehighest manufacturing yields of biological products.

The present invention removes all structural and technical hurdles inmaking use of the technology related to flexible bags being used forlarge scale bioreactors; a pressurized bag it within a hard-walledcylindrical support relieving any stress on the seam of the bag and thisallows use of construction of bioreactors that can easily containthousands of liters of nutrient media compared to the prior art thatinevitably restricts such volumes to only a few hundred liters.

Although the present bag has been described and illustrated in detail,it is to be clearly understood that this is done by way of illustrationand example only and is not to be taken by way of limitation. The scopeis to be limited only by the terms of the appended claims.

What is claimed is:
 1. A mixing device comprising: a) a cylindrical support with an inner surface configured to immovably retain a single-use flexible container; b) a single-use flexible container capable of containing a liquid comprising: i) A flexible septum with an inner surface immovably positioned within the flexible container and defining a right chamber and a left chamber, the septum comprising a plurality of pores with a variable density over the surface of the septum; ii) At least one liquid inlet; iii) At least one liquid outlet; iv) At least one gas inlet in fluid communication with a source of compressed gas; v) At least one gas outlet capable of controlling the rate of flow of gas; vi) at least one sensor to measure the temperature of a liquid in the flexible container; and vii) A heater and/or a cooling element for controlling the temperature of the flexible container; c) a shaft for pivoting the cylindrical support along its circular axis between −110 to +110 degrees; and wherein, the flexible container is immovably fixed to the inner surface of the cylindrical support.
 2. The mixing device of claim 1, wherein the pore size of the plurality of pores in the porous septum ranges 1 μm to 10,000 μm.
 3. The mixing device of claim 1, wherein the pore size of the plurality of pores in the porous septum ranges 100 μm to 10,000 μm.
 4. The mixing device of claim 1, wherein the surface of the flexible septum comprises 3-5 times higher density of pores along the middle of the horizontal axis.
 5. The mixing device of claim 1, further comprising a liquid wherein the liquid is comprised of a mixture of a plurality of liquids, a mixture of a plurality of liquids and a plurality of gases or a mixture of a plurality of solids and a plurality of liquids.
 6. The mixing device according to claim 1, wherein the flexible container is substantially cylindrical, ovoid, cuboid, round, rectangular or square in shape.
 7. The mixing device according to claim 1, wherein the flexible container is a flexible bag, and wherein the internal portion of the flexible container is comprised of a biocompatible material.
 8. The mixing device according to claim 1, further comprising a sensor capable of measuring the pressure inside the flexible container.
 9. The mixing device of claim 1, further comprising a controller capable of controlling the frequency and the degree of pivoting of the cylinder, and the temperature of a liquid in the flexible container.
 10. The mixing device of claim 8, further comprising a controller capable of controlling the pressure inside the flexible container. 